CN113416196A - benzothiadiazole-TB compound and synthesis method and application thereof - Google Patents

Abstract

The invention provides a benzothiadiazole-TB compound, a synthesis method and an application thereof, wherein the structural general formula of the benzothiadiazole-TB compound is shown as a formula A:

wherein, R is one of hydrogen, alkoxy, hydroxyl and alkyl. The benzothiadiazole-TB compound has large Stokes displacement, excellent luminescence performance, excellent solid luminescence and potential of becoming an excellent OLED material; has wide pH application range, and can be applied to human physiological environmentPerforming the following steps; the probe has effective and good response to viscosity and can become a fluorescent probe with viscosity response; for Fe³⁺Has identification ability and is expected to be excellent Fe³⁺A fluorescent probe; the PDT photosensitizer has a good PDT effect, can be used for dyeing and imaging A549 cells, expands the types of the photosensitizer, and provides a new idea for synthesizing imaging-guided PDT photosensitizer.

Description

benzothiadiazole-TB compound and synthesis method and application thereof

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to synthesis of a benzothiadiazole-TB derivative with excellent optical performance and application of the benzothiadiazole-TB derivative in metal ion recognition, photodynamic therapy and biological imaging.

Background

Photodynamic Therapy (PDT) is a new method for treating diseases such as tumors by using photosensitive drugs and laser activation. Compared with the traditional method, the method has the advantages of low side effect, minimal invasion, no obvious drug resistance, high tumor destruction selectivity, easy combination with other therapies and the like. Photodynamic therapy (PDT) has better space-time accuracy and effectiveness in clinical research and treatment, and becomes an important emerging means for accurate treatment of clinical tumors. With the rapid development of PDT, people have increasingly high requirements on the variety of photosensitizers, the efficiency of photosensitizers, and the like. Aiming at the problems of visible absorption, poor penetrability and the like of the conventional photosensitizer for PDT, more and more novel high-efficiency photosensitizers are developed. However, photosensitizers based on the TB skeleton are still in the primary stage, and there is only one relevant report.

Fluorescence imaging has extremely high time and space resolution, is an effective research means for understanding cell metabolism, observing cell morphology and tracking physiological changes of organisms, and has been widely applied to multiple fields of modern medicine industry, such as biotechnology, clinical diagnosis and the like, as a powerful tool for detecting target molecules and life processes in a life system. The optical properties of fluorescent materials play a crucial role in cellular or in vivo fluorescence imaging applications.

Intracellular viscosity is an important influence factor of chemical signal transmission, intermolecular interaction and material exchange, and can be used as a reference index for diagnosing certain diseases, such as Alzheimer's disease, cell malignancy, atherosclerosis and the like, so that detection of viscosity change at a cell level is very important for maintaining normal life activities. The fluorescent probe has the advantages of convenient use, high sensitivity, high response speed and the like, and is widely applied to detecting the change of cell viscosity.

2,1, 3-Benzothiadiazole (BTD) is an electron-deficient group containing nitrogen and sulfur atoms with planar aromaticity, and the pi-plane expansion of the group is favorable for enhancing the electron flow between A and D, simultaneously increasing the intermolecular interaction and providing a well-ordered crystal structure. BTD can be used as an electron acceptor to enable the material to keep high luminous efficiency; has a larger T₂-T₁The energy is extremely poor, the conversion rate in a triplet exciton can be reduced, the energy gap is adjusted, and the optical performance is excellent; the structure is easy to modify, and the method is widely applied to the fields of fluorescent probes, ion recognition, organic light-emitting diodes, solar cells and the like.

Therefore, three benzothiadiazole-TB compounds are designed and synthesized by introducing a benzothiadiazole group into a TB skeleton, and are applied to the fields of metal ion identification, photodynamic therapy, biological imaging and the like.

Disclosure of Invention

The invention aims to provide a benzothiadiazole-TB compound, a synthesis method and application thereof

The base compound is applied to the fields of metal ion identification, photodynamic therapy, biological imaging and the like.

A benzothiadiazole-TB compound has a structural general formula shown as a formula A:

wherein, R is one of hydrogen, alkoxy, hydroxyl and alkyl.

Preferably, the structural formula of the benzothiadiazole-TB compound is one of the following:

a synthetic method of a benzothiadiazole-TB compound comprises the following steps:

(1) reacting the compound 1 or 2 with paraformaldehyde to obtain an intermediate 3 or 4, wherein the reaction formula is as follows:

(2) reacting the intermediate 3 or 4 with trimethyl borate to obtain an intermediate 5 or 6, wherein the reaction formula is as follows:

(3) the intermediate 5 or 6 and 2, 7-dibromo-benzothiadiazole (7) are subjected to coupling reaction to obtain a compound 8 or 9, wherein the reaction formula is as follows:

(4) hydrolysis of compound 9 affords compound 10, according to the following reaction scheme:

the benzothiadiazole-TB compound is applied as a photosensitizer for photodynamic therapy.

The benzothiadiazole-TB compound is applied to biological imaging as a fluorescent material.

The benzothiadiazole-TB compound is used as Fe³⁺The application of the fluorescent probe in metal ion recognition.

Has the advantages that: the benzothiadiazole-

base compoundsThe product has the following advantages:

(1) the synthesis method is simple, and the post-treatment is convenient;

(2) the material has large Stokes shift, excellent luminescence property, excellent solid-state luminescence and potential of becoming an excellent OLED material;

(3) has wide pH application range and can be applied to human physiological environment;

(4) the probe has effective and good response to viscosity and can become a fluorescent probe with viscosity response;

(5) for Fe³⁺Has identification ability and is expected to be excellent Fe³⁺A fluorescent probe;

(6) the PDT photosensitizer has a good PDT effect, can be used for dyeing and imaging A549 cells, expands the types of the photosensitizer, and provides a new idea for synthesizing imaging-guided PDT photosensitizer.

Drawings

FIG. 1 is a drawing of the product of Compound 8 in the examples¹H NMR spectrum;

FIG. 2 is a drawing of the product, Compound 8, of the example¹³C NMR spectrum;

FIG. 3 is a drawing of the product of example, Compound 9¹H NMR spectrum;

FIG. 4 is a drawing of the product, Compound 9, of the example¹³C NMR spectrum;

FIG. 5 is a drawing of the product Compound 10 of the example¹H NMR spectrum;

FIG. 6 is a drawing of the product Compound 10 of the example¹³C NMR spectrum;

FIG. 7a is a UV absorption spectrum of Compound 8 in different solvents;

FIG. 7b is the fluorescence emission spectra of Compound 8 in different solvents;

FIG. 7c is a UV absorption spectrum of Compound 9 in different solvents;

FIG. 7d is the fluorescence emission spectra of Compound 9 in different solvents;

FIG. 7e is a UV absorption spectrum of Compound 10 in different solvents;

FIG. 7f is a fluorescence emission spectrum of Compound 10 in different solvents;

FIG. 8 is a fluorescence emission spectrum (a) and a line graph (b) of Compound 9 at different viscosities;

FIG. 9 is a fluorescence emission spectrum (a) and a line graph (b) of Compound 9 at different pH;

FIG. 10 shows different concentrations of Fe³⁺Fluorescence emission spectrum (a) and line graph (b) of compound 9 in the presence;

FIG. 11 shows different concentrations of Fe³⁺Fluorescence emission spectrum (a) and standard curve (b) of compound 9 in the presence;

FIG. 12 is the compound 9-Fe³⁺The Job's curve of the system;

FIG. 13 shows compound 9 with Fe³⁺Possible coordination modes;

FIG. 14 is a fluorescence micrograph of 9 pairs of A549 cells in the absence of illumination (L-) and illumination (L +).

Detailed Description

The invention designs and synthesizes benzothiadiazole-doped drug by introducing benzothiadiazole group on TB skeleton

The base compound is applied to the fields of metal ion identification, photodynamic therapy, biological imaging and the like.

Benzothiadiazole-

The general structural formula of the base compound is shown as formula A:

wherein, R is one of hydrogen, alkoxy, hydroxyl and alkyl.

TABLE 1 exemplary Compounds of the Compounds of formula A

The present invention will be further described with reference to the following examples.

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

Examples

In the embodiment, para-bromoaniline, paraformaldehyde, n-butyl lithium, 4, 7-dibromo-benzothiadiazole and the like are used as raw materials and are prepared through coupling reaction. The method comprises the following steps:

reacting the compound 1 or 2 with paraformaldehyde to obtain an intermediate 3 or 4, reacting the intermediate 3 or 4 with trimethyl borate to obtain an intermediate 5 or 6, and carrying out coupling reaction on the intermediate 5 or 6 and 2, 7-dibromo-benzothiadiazole (7) to obtain a compound 8 or 9, and further hydrolyzing the compound 9 to obtain a compound 10.

By the above synthetic method, the compounds of the following examples were prepared:

(1) 4-bromoaniline 1(50.0mmol) and paraformaldehyde (100.0mmol) were sequentially added to a 200.0mL round-bottomed flask, the flask was placed in a low-temperature bath, the temperature was adjusted to-15 ℃, trifluoroacetic acid (100.0mL, dropwise addition completed in about 30 min) was slowly added to the flask with stirring, and then the mixture was reacted at room temperature for 7 days. After completion of the reaction (TLC trace), the mixture was poured into ice water, adjusted to pH 9-10 with ammonia, cooled to room temperature, extracted with dichloromethane (50.0mL × 3), and spin dried to give the crude product. Adding acetone, heating until the crude product is completely dissolved, recrystallizing at room temperature, filtering, and washing with acetone to obtain intermediate 3.

(2) 4-bromo-3-methoxyaniline 2(50.0mmol) and paraformaldehyde (100.0mmol) were sequentially added to a 200.0mL round-bottomed flask, the flask was placed in a cryotank, the temperature was adjusted to-15 ℃, trifluoroacetic acid (100.0mL, about 30min after completion of dropwise addition) was slowly added to the flask with stirring, and then the reaction was carried out at room temperature for 7 days. After completion of the reaction (TLC trace), the mixture was poured into ice water, adjusted to pH 9-10 with ammonia, cooled to room temperature, filtered with suction, and washed three times with ethanol to give intermediate 4.

Equation 1TB-Br and TB-OCH₃Synthesis of-Br

(3) Adding 3 or 4(5.0mmol) into a 100mL round-bottom flask, vacuumizing for three times, placing the flask in a low-temperature tank, adjusting the temperature to-78 ℃, adding 20.0mL of anhydrous tetrahydrofuran into the flask under stirring, dropwise adding 2.5mL of n-butyl lithium, reacting for 1h under the protection of argon, dropwise adding 0.6mL of trimethyl borate, and then placing the flask at room temperature for reacting for 4 h. TLC trace to completion of reaction, dichloromethane extraction (30.0 mL. times.3) and spin-drying to give crude product. The crude product is purified by column chromatography (V)_PE:V_EA1) to give intermediate 5 (65%) or 6 (70%).

Synthesis of intermediates 5 and 6 of equation 2

(4) 5 or 6(2.0mmol), 4, 7-dibromo-benzothiadiazole 7(4.8mmol) and tetrakis (triphenylphosphine) palladium (20% mmol,0.06g) were sequentially added to a 100mL round-bottomed flask, 35.0mL of anhydrous toluene was added under argon protection, and then potassium carbonate (0.53g) was added, followed by reaction at 108 ℃ for 12 hours. After completion of the reaction (TLC chase), it was cooled to room temperature, extracted with dichloromethane (10.0 mL. times.3), the organic phase was dried over anhydrous sodium sulfate, and dried to give a crude product which was purified by column chromatography (V)_DCM:V_EA100:1) to obtain compound 8 (65%) or 9 (70%).

2, 8-bis (7-bromobenzo [ c ] [1,2,5] thiadiazol-4-yl) -6H,12H-5, 11-dibenzo [ b, f ] [1,5] diazocine (8)

¹H NMR(400MHz,CDCl₃)δ7.88(d,J＝7.6Hz,2H,Ar-H),7.75(d,J＝8.4Hz,2H,Ar-H),7.53(s,2H,Ar-H),7.48(d,J＝7.6Hz,2H,Ar-H),7.40(d,J＝8.0Hz,2H,Ar-H),4.93(d,J＝16.4Hz,2H,-CH₂-bridge),4.51-4.40(m,4H,TB-CH _2*2).¹³C NMR(100MHz,CDCl₃)δ153.9,153.1,132.4,128.7,128.1,127.9,125.5,66.9,58.6,29.8.

7,7' - (3, 9-dimethoxy-6H, 12H-5, 11-dibenzo [ b, f ] [1,5] diazocine-2, 8-diyl) bis (4-bromobenzo [ c ] [1,2,5] thiadiazole) (9)

¹H NMR(400MHz,CDCl₃)δ7.82(d,J＝7.2Hz,1H,Ar-H),7.40(d,J＝7.6Hz,1H,Ar-H),7.02(s,1H,Ar-H),6.85-6.61(m,5H,Ar-H),4.73-4.68(m,2H,-CH₂-bridge),4.32-4.18(m,4H,TB-CH_2*2),3.73(d,J＝8.8Hz,6H,-OCH₃*2).¹³C NMR(100MHz,CDCl₃)δ159.1,156.3,153.9,153.4,149.7,149.0,132.0,131.2,130.2,129.7,127.8,122.2,120.0,119.8,112.8,111.3,109.6,107.9,66.8,58.2,58.1,55.8,55.4.

Equation 3 Synthesis of Compounds 8 and 9

(5) And (3) putting the compound 9(2.0mmol) in a 50mL round-bottom flask, vacuumizing for three times, placing the flask in a low-temperature tank, adjusting to-15 ℃, adding 15.0mL of anhydrous dichloromethane, dropwise adding boron tribromide, moving the reaction bottle to room temperature after dropwise addition, and continuing to react for 12 hours under the protection of argon. After completion of the reaction (TLC run), water was added for quenching, dichloromethane was extracted (10.0mL × 3), and the organic phase was dried over anhydrous sodium sulfate to give compound 10 (75%) after spin-drying.

Equation 4 Synthesis of Compound 10

2, 8-bis (7-bromobenzo [ c ] [1,2,5] thiadiazol-4-yl) -6H,12H-5, 11-dibenzo [ b, f ] [1,5] diazocine-3, 9-diol (10)

¹H NMR(400MHz,DMSO-d₆)δ8.06(d,J＝7.6Hz,1H,Ar-H),7.58(d,J＝7.6Hz,1H,Ar-H),7.22(s,1H,Ar-H),7.03(d,J＝8.4Hz 1H,Ar-H),6.96(s,1H,Ar-H),6.79-6.71(m,3H,Ar-H),4.88-4.80(m,4H,TB-CH_2*2),4.32(dd,J₁＝6.0Hz,J₂＝16.0Hz,2H,-CH₂-bridge).¹³C NMR(100MHz,DMSO-d₆)δ157.6,155.1,153.7,153.1,132.7,130.8,130.7,130.4,128.9,112.4,111.3,110.7,66.3,56.9

Optical Performance testing

The solvating effect of the compounds of the invention was tested, and the specific experimental protocol was as follows:

compounds 8, 9 and 10 were formulated with eight solvents, Dichloromethane (DCM), Tetrahydrofuran (THF), methanol (MeOH), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), Toluene (Toluene), N, N-Dimethylformamide (DMF), N-Hexane (N-Hexane) to a concentration of 1X 10, respectively^-5And (3) testing the ultraviolet absorption spectrum and the fluorescence emission spectrum of the working solution in mol/L. As shown in fig. 7 a-7 f.

As can be seen from FIGS. 7a to 7f, the relative fluorescence intensity of compound A in medium and small polar solvents is strong, and lambda is_emA significant red shift occurs and a blue shift occurs in highly polar solvents. The ultraviolet absorption peak appears at about 290nm and belongs to pi-pi on an aromatic ring^*B-band absorption due to the transition; its UV absorption at 390nm is attributed to n-pi on the heteroatom^*R-band absorption caused by the transition.

The ultraviolet absorption and fluorescence emission spectra and the solid state fluorescence emission spectra of the compound in DCM solution were tested, and the specific experimental protocol was as follows:

weighing 10^-5mol of compounds 3, 4, 5, 6, 7, 8, 9 and 10 were made up to a concentration of 1X 10 with DCM solution^-5And (5) testing ultraviolet absorption, fluorescence emission and solid state fluorescence emission spectra of the sample at mol/L.

3. The spectral data for 4,7, 8, 9 and 10 are shown in table 2.

As shown in table 2, 10 has the largest molar absorption coefficient, probably because the substituent is hydroxyl, the electron cloud density is the largest, and the transition is most likely to occur under light excitation; in addition, hydroxyl is easy to form intramolecular chelate hydrogen bonds with adjacent thiadiazole heterocycles, so that the molecular rigidity is increased and the electron conjugation degree is improved. The substituent of 9 is methoxy group, which has certain electron-withdrawing induction effect, so that the absorbance is less than 10 of the substituent which is hydroxyl.

Compared with the raw materials 3, 4 and 7, the absorbance of the product 8-10 is enhanced, which shows that the electron cloud density of the compound is improved after the compound is connected with a TB skeleton.

TABLE 2 spectroscopic Data (DCM) for compounds 3, 4,7, 8, 9 and 10

^aUltraviolet absorption wavelength in solution (slit is 2.5/5 nm);^bmolar extinction coefficient ε is A/bC, unit is 1 × 10⁵L·mol^-1·cm^-1；^cFluorescence emission wavelength in solution;^dstokes shift in solution;^erelative fluorescence quantum yield (reference: quinine sulfate);^ffluorescence intensity in L.mol^-1·cm^-1；^gSolid state excitation wavelength (slit 2.5/2.5 nm);^ha solid state fluorescence emission wavelength;ⁱsolid-state Stokes shift. "-" indicates that the intensity was too low to test.

The fluorescence properties of the product compared to starting materials 4 and 7 have the following changes:

(1) solutions and solid lambda of Compounds 8 to 10_emObvious red shift occurs, and the displacement of solution and solid Stokes is obviously increased;

(2) the relative fluorescence quantum yield of the compound 8 and 9 solutions is obviously increased, and the luminous brightness is obviously enhanced;

(3) the solution of compound 10 showed insignificant changes in the relative fluorescence quantum yield and the luminescence brightness.

This is probably due to the fact that compounds 8 and 9 have highly distorted structures, which hinder the rotation of their sub-molecular radicals (RIR mechanism), thereby enhancing the solid-state emission intensity; while the compound 10 forms intramolecular hydrogen bonds in the solid state, which causes OH-O stretching vibration, increases non-radiative efficiency and results in weaker solid-state fluorescence emission.

The above results show that combining the TB skeleton with the benzothiadiazole group can amplify the advantages of the TB skeleton and the benzothiadiazole group in the luminescence property, and the method is a new way for obtaining products with excellent luminescence property.

Viscosity response test

Taking compound 9 as an example, compound 9 was formulated to a concentration of 1X 10 using methanol as a solvent^-4mol·L^-1The working solution of (1). Taking 10mL volumetric flasks, adding 0-9mL glycerol, respectively, measuring 1mL working solution in the volumetric flasks, and diluting with methanol to constant volume to make the concentration of 1 × 10^-5mol·L^-1And fluorescence emission spectrum (methanol: glycerol are 1: 9-10: 0 in sequence) (lambda) is measured_ex270nm, slit: 2.5/5nm) (FIG. 8).

As can be seen from fig. 8, the fluorescence intensity of compound 9 increased with the increase in viscosity. This is probably due to the fact that in low viscosity, the degree of intramolecular rotation is high and the fluorescence intensity is reduced; with the increase of viscosity, the movement of the sub-molecular radicals is hindered, and the molecules release energy mainly in a radiation mode, so that the fluorescence intensity is increased (RIR mechanism). This result demonstrates the potential of compound 9 to be a viscosity-responsive fluorescent probe.

pH response test

Taking compound 9 as an example, its response to pH was tested. Compound 9 was formulated at a concentration of 1X 10 using THF as the solvent^-4mol·L^-1Respectively weighing 1mL of the working solution into 10mL volumetric flasks, adding 1mL of buffer solution with pH value of 2-10 (citric acid/disodium hydrogen phosphate system is selected when pH value is 2-8, and sodium bicarbonate/sodium carbonate system is selected when pH value is 9-10), and diluting with THF to constant volume to make concentration 1 × 10^-5mol L^-1The fluorescence emission spectrum (lambda) is measured_ex280nm, slit: 2.5/5nm) as shown in fig. 9.

As can be seen from FIG. 9, when the pH of the solution is 2-10, the fluorescence emission spectrum has no significant change, indicating that 9 has a wide pH application range and can be applied to human physiological environment.

Ion detection test

9(0.0071g, 1X 10) was weighed out^-5mol) in a 100mL volumetric flask, and DMSO is used for constant volume to prepare the product with the concentration of 1 × 10^{- 4}mol·L^-1The working fluid of (1). 10mL volumetric flasks were each charged with 1mL of 1X 10^-2mol L^-1Fe (b) of³⁺、Na⁺、K⁺、Cu²⁺、Zr⁴⁺、Al³⁺、PO₄ ^3-、F^-Cys, GSH, Hcy and thioglycol solution, adding 1mL PBS buffer solution, measuring 1mL working solution in the 10mL volumetric flask, diluting with DMSO to constant volume, and measuring fluorescence emission spectrum (lambda)_ex280nm, slit: 5/10 nm).

10(0.0068g, 1X 10) were likewise tested by the methods described above^-5mol) with Fe³⁺、Na⁺、K⁺、Cu²⁺、Zr⁴⁺、Al³⁺、PO₄ ^3-、F^-Fluorescence emission spectrum (λ)_ex290nm, slit: 5/10 nm).

The fluorescence intensity of the solution of the compound 9 or 10 without addition of metal ions is denoted as I₀The fluorescence intensity after adding the metal ion solution is recorded as I, the fluorescence intensity change rate of the compound after adding the metal ion is recorded as eta, and the identification efficiency eta is (I-I)₀)/I₀X 100%. The changes of fluorescence intensity of the compounds 9 and 10 after the action with different ions and biological thiol are shown in tables 3 and 4.

TABLE 3 Effect of Compound 9 with different Ionic, biological thiols

^aThe compound has the fluorescence intensity change rate after adding metal ions, wherein eta is (I-I)₀)/I₀X 100%. ' "/" indicates none.

TABLE 4 Effect of Compound 10 with different ions

^aFluorescence intensity of compound after adding metal ionRate of change, eta ═ I (I-I)₀)/I₀X 100%. "/" indicates none.

As can be seen from tables 3 and 4, the ionic radius has no correlation with the recognition efficiency. Compounds 9 and 10 with Fe³⁺After action, its lambda_emAll showed significant red shift indicating compounds 9 and 10 with Fe³⁺A coordination reaction may occur.

Further study of compound 9 on Fe³⁺Identification of (2): 1mL of compound 9 working solution (1X 10) was measured out in this order^{- 4}mol·L^-1) 1mL of PBS buffer solution and 7mL of DMSO in a 10mL volumetric flask, and then measured from a concentration of 1X 10^-2mol·L^-1Fe (b) of³⁺Respectively adding solutions with different volumes into the volumetric flask, diluting with redistilled water to constant volume, and making into the solution with concentration of 1 × 10^-6-1×10^-3mol·L^-1To observe the influence of (lambda) on the fluorescence of Compound 9_ex280nm, slit: 5/10nm) (FIG. 10).

As shown in FIG. 10, in Fe³⁺The concentration is 5X 10^-6-2×10^-4In this range, the fluorescence intensity of compound 9 showed a sharp decrease, while as the concentration was further increased, the fluorescence intensity varied slowly until complete quenching.

The compound 9-Fe was subsequently explored³⁺System standard curve: (FIG. 11, R)²1.00), compound 9 vs Fe was calculated³⁺Limit of detection (LOD ═ 3 δ)_blankK, wherein δ_blankAnd k is the standard deviation of the blank solution and the slope of the calibration curve, respectively). LOD of 9.6X 10^-6mol·L^-1Indicating that Compound 9 has the development of Fe³⁺Potential of fluorescent probes.

Identification of Fe for exploration 9³⁺The reason for (1) is 9 and Fe³⁺The total concentration was constant, ranging from 1:9 to 9:1, varying 9 and Fe³⁺The fluorescence intensity of each solution was measured, and a Job's curve (. lamda.) was plotted_ex280nm, slit: 5/10nm, concentration of 1X 10^-5mol·L^-1) (FIG. 12). As can be seen from FIG. 12, 9 is in contact with Fe³⁺The fluorescence intensity shows an inflection point at a molar ratio of 3:7Probably due to 9 and Fe³⁺A complex is formed with a coordination ratio of about 1: 2. Thus, 9 and Fe are presumed³⁺The possible coordination patterns are shown in FIG. 13.

Cytotoxicity test

The cytotoxicity of the raw material 7 and the compounds 8, 9 and 10 on human bronchial epithelial-like (HBE) cells was examined by MTT using a model of HBE cells. HBE cells were seeded in 96-well plates (1X 10)^-5one/mL), 100. mu.L of medium was added to each well, CO at 37 ℃₂After 24h incubation in the incubator, compounds 7, 8, 9 and 10 at different concentrations were added to the seeded cells and incubated for 24 h. The plates were then washed 3 times with PBS buffer and 10. mu.L of MTT solution was added to each well for an additional 4h incubation. The medium in the wells was removed, 150 μ L of DMSO was added to each well to dissolve the blue-violet formazan (Formazam) crystals in the cells, and the cells were placed on a shaker and shaken at low speed for 5-7min to dissolve the crystalline material thoroughly. Finally, an enzyme-linked immunosorbent assay (ELISA) detector is adopted to measure the absorbance values of each hole at 560nm and 670 nm. Cytotoxicity was calculated by the following formula:

％viability＝[∑(A_i/A₀×100)/n]

in the formula A_iRespectively the absorbance values of the compounds with different concentrations; a. the₀Mean absorbance values for control wells without added compound; n (═ 3) represents three replicates.

The MTT method is adopted to detect the biological toxicity of the compound 9 to Human Bronchial Epithelial (HBE) cells and the dark toxicity and the phototoxicity to human non-small lung cancer (A549) cells. The light source was 423nm, the control was not exposed to light, and absorbance values at 560nm and 670nm were measured for each well using an enzyme linked immunosorbent assay.

TABLE 5 half-maximal Inhibition (IC) of Compound 9 on both cells₅₀)

The half inhibition rate of compound 9 on HBE and A549 cells is shown in Table 5, and the results show that compound 9 has extremely low dark toxicity and high phototoxicity on A549 cells, the PDT efficiency is high, but the phototoxicity on HBE cells is also high, and further structural modification is needed to reduce the phototoxicity.

Cell imaging assay

A549 cells are inoculated into a 96 micro-well plate and cultured for 24 h. The cells were then incubated with different concentrations (6.25, 12.50. mu. mol/L) of Compound 9 for an additional 24h, half with 2h of light and half without light. The medium was changed to a medium containing calcein (CA, 100. mu.g/mL) for further 24h to stain live and dead cells. The plate was then washed 3 times with PBS buffer and incubated for 4h with MTT solution. The medium was removed and DMSO was added and shaken at low speed for 5-7 min. Finally, the cells were analyzed using fluorescence microscopy.

The effect of product 9 on photodynamic therapy of a549 cells in vitro was directly observed by live/dead cell staining (figure 14). Viable cells can be stained with Calcein (CA) as a green signal.

As shown in fig. 14, a549 cells (control group) not treated with compound 9 had no significant change in morphology before and after light irradiation. In the absence of light, the concentrations were 6.25 and 12.50. mu. mol. multidot.L^-1None of the dark groups treated with compound 9 caused significant cell death, indicating that compound 9 had no dark toxicity; under the condition of illumination, obvious cell death is observed in fluorescence micrographs after treatment of the compound 9 at two concentrations, the cell morphology is damaged, and only yellow-green luminescence of the compound 9 can be observed, which indicates that the compound 9 has better phototoxicity on A549 cells, and the result is mutually verified with the result in the experimental analysis of in vitro photodynamic therapy.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims (6)

1. A benzothiadiazole-TB compound is characterized in that: the general structural formula is shown as formula A:

wherein, R is one of hydrogen, alkoxy, hydroxyl and alkyl.

2. The benzothiadiazole-TB-like compound of claim 1, wherein: the structural formula is one of the following:

3. a method for synthesizing the benzothiadiazole-TB compound of claim 1, which is characterized by: the method comprises the following steps:

(1) reacting the compound 1 or 2 with paraformaldehyde to obtain an intermediate 3 or 4, wherein the reaction formula is as follows:

(2) reacting the intermediate 3 or 4 with trimethyl borate to obtain an intermediate 5 or 6, wherein the reaction formula is as follows:

(3) the intermediate 5 or 6 and 2, 7-dibromo-benzothiadiazole (7) are subjected to coupling reaction to obtain a compound 8 or 9, wherein the reaction formula is as follows:

(4) hydrolysis of compound 9 affords compound 10, according to the following reaction scheme:

4. use of the benzothiadiazole-TB-type compound of claim 1 as a photosensitizer for photodynamic therapy.

5. The benzothiadiazole-TB compound of claim 1, as a fluorescent material for use in biological imaging.

6. benzothiadiazole-TB-type compounds as described in claim 1 as Fe³⁺The application of the fluorescent probe in metal ion recognition.

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